How do cells respond to mechanical stimulation? We know that mechanosensitive ion channels (MSCs) are in all cells in all kingdoms, yet our biophysical understanding of how these channels function as transducers in the eukaryotic world lags in comparison to voltage and ligand-gated channels. Over the past decade a wealth of information derived from genetics and electrophysiological analysis of mechanical transduction has emerged from model systems such as C. elegans and bacteria. But the biophysics of mechanical gating at the molecular level in eukaryotic systems, such as direct measures of the forces needed to open channels have been confined to measuring endogenous channel activity rather than cloned channels. Two years ago a new channel family called Piezo was identified that is mechanically gated and has many, if not all, the properties of the endogenous channels we and others have studied for years. The Piezo family with nearly 2500 residues and 25-32 transmembrane domains bears no relationship to other channels (such as TRPs). They activate with membrane tension and inactivate with time, and much in parallel with endogenous channels, inactivation can be lost changing the channels from phasic to tonic sensors. The discovery of this channel family provided us with new opportunities to answer specific biophysical questions related to the gating of mechanical channels. The importance of this channel is underscored by the recent study showing single mutations to Piezo1 in erythrocytes leads to a condition called Xerocytosis, an inability to regulate cell volume. This laboratory has a long history of studying MSCs and their integration into cell mechanics. We discovered MSCs, developed the standard methods of study, the software and instrumentation to study them, characterized their biophysics, found the specific inhibitors and demonstrated their relevance to disease. We will study Piezo channels using electrophysiology at single and multichannel resolution in cells and reconstituted liposomes, generic expression systems and differentiated cells, light microscopy, molecular biology and mathematical analysis. The basic aims include Characterize ionic selectivity for monovalents and divalents. Characterize inactivation/activation using kinetic analysis of mutant channels. Characterize the cytoskeletal and membrane proteins that are involved in the cooperative loss of inactivation and responsible for creating mechanical domains. Visualize the mechanical domain structures in cells using high resolution light microscopy. Measure stress in patches addressing bilayer stress using dynamic capacitance and calibrated bacterial MSC gating standards, and cortical stress using high resolution optical microscopy and genetically coded FRET based probes of stress in specific cytoskeleton proteins.